Comments on Cui Q-Q et al: "Hippocampal CD 39/ENTPD 1 promotes mouse depression-like behavior ".

Comment on Hippocampal CD 39/ENTPD 1 promotes mouse depression-like behavior through hydrolyzing extracellular ATP by Cui et al.

Hyperpolarization of cis-15 N2 -Azobenzene by Parahydrogen at Ultralow Magnetic Fields*.

The development of nuclear spins hyperpolarization, and the search for molecules that can be efficiently hyperpolarized is an active area in nuclear magnetic resonance. In this work we present a detailed study of SABRE SHEATH (signal amplification by reversible exchange in shield enabled alignment transfer to heteronuclei) experiments on 15 N2 -azobenzene. In SABRE SHEATH experiments the nuclear spins of the target are hyperpolarized through transfer of spin polarization from parahydrogen at ultralow fields during a reversible chemical process. Azobenzene exists in two isomers, trans and cis. We show that all nuclear spins in cis-azobenzene can be efficiently hyperpolarized by SABRE at suitable magnetic fields. Enhancement factors (relative to 9.4 T) reach up to 3000 for 15 N spins and up to 30 for the 1 H spins. We compare two approaches to observe either hyperpolarized magnetization of 15 N/1 H spins, or hyperpolarized singlet order of the 15 N spin pair. The results presented here will be useful for further experiments in which hyperpolarized cis-15 N2 -azobenzene is switched by light to trans-15 N2 -azobenzene for storing the produced hyperpolarization in the long-lived spin state of the 15 N pair of trans-15 N2 -azobenzene.

Ectonucleoside triphosphate diphosphohydrolases and ecto-5'-nucleotidase in purinergic signaling: how the field developed and where we are now.

Geoffrey Burnstock will be remembered as the scientist who set up an entirely new field of intercellular communication, signaling via nucleotides. The signaling cascades involved in purinergic signaling include intracellular storage of nucleotides, nucleotide release, extracellular hydrolysis, and the effect of the released compounds or their hydrolysis products on target tissues via specific receptor systems. In this context ectonucleotidases play several roles. They inactivate released and physiologically active nucleotides, produce physiologically active hydrolysis products, and facilitate nucleoside recycling. This review briefly highlights the development of our knowledge of two types of enzymes involved in extracellular nucleotide hydrolysis and thus purinergic signaling, the ectonucleoside triphosphate diphosphohydrolases, and ecto-5'-nucleotidase.

Identification of adenine-N9-(methoxy)ethyl-ß-bisphosphonate as NPP1 inhibitor attenuates NPPase activity in human osteoarthritic chondrocytes.

Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate) dihydrate deposition (CPPD) in cartilage, resulting in a degenerative joint disease that today lacks a cure. Here, we targeted the identification of novel NPP1 inhibitors as potential therapeutic agents for CPPD deposition disease. Specifically, we synthesized novel analogs of AMP (NPP1 reaction product) and ADP (NPP1 inhibitor). These derivatives incorporate several chemical modifications of the natural nucleotides including (1) a methylene group replacing the Pα,ß-bridging oxygen atom to provide metabolic resistance, (2) sulfonate group(s) replacing phosphonate(s) to improve binding to NPP1's catalytic zinc ions, (3) an acyclic nucleotide analog to allow flexible binding in the NPP1 catalytic site, and (4) a benzimidazole base replacing adenine. Among the investigated compounds, adenine-N9-(methoxy)ethyl-ß-bisphosphonate, 10, was identified as an NPP1 inhibitor (Ki 16.3 µM vs. the artificial substrate p-nitrophenyl thymidine-5'-monophosphate (p-Nph-5'-TMP), and 9.60 µM vs. the natural substrate, ATP). Compound 10 was selective for NPP1 vs. human NPP3, human CD39, and tissue non-specific alkaline phosphatase (TNAP), but also inhibited human CD73 (Ki 12.6 µM). Thus, 10 is a dual NPP1/CD73 inhibitor, which could not only be of interest for treating CPPD deposition disease and calcific aortic valve disease but may also be considered for the immunotherapy of cancer. Compound 10 proved to be a promising inhibitor, which almost completely reduces NPPase activity in human osteoarthritic chondrocytes at a concentration of 100 µM.

Correction to: Identification of adenine-N9-(methoxy)ethyl-ß-bisphosphonate as NPP1 inhibitor attenuates NPPase activity in human osteoarthritic chondrocytes.

The original version of the article unfortunately contained an error.

Adenine-(methoxy)-ethoxy-Pα,α-dithio-triphosphate inhibits pathologic calcium pyrophosphate deposition in osteoarthritic human chondrocytes.

Nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) inhibitors have been suggested as a potential treatment for calcium pyrophosphate dihydrate (CPPD) deposition disease. Here, we targeted the development of improved NPP1 inhibitors based on acyclic mimics of Pα,α-phosphorodithioate-substituted adenine nucleotides, 7-10. The latter were obtained in a facile two-step synthesis from adenine-(methoxy)ethanol. Among analogs 7-10, adenine-(methoxy)ethoxy-Pα,α-dithio-triphosphate, 8, was the most potent NPP1 inhibitor both with purified enzyme (IC50 0.645 µM) and in osteoarthritic human chondrocytes (IC50 0.033 µM). Furthermore, it efficaciously (10-fold vs. control) inhibited ATP-induced CPPD in human articular chondrocytes. Importantly, 8 was a highly selective NPP1 inhibitor which showed only minor inhibition of NPP3, CD39 and CD73, and did not inhibit TNAP (tissue nonspecific alkaline phosphatase) activity in human chondrocytes. Furthermore, 8 did not activate P2Y1,2,6 receptors. Analog 8 was not toxic to cultured chondrocytes at 100 µM. Therefore, 8 may be suitable for further development as a drug candidate for the treatment of CPPD arthritis and other NPP1-related diseases.

Constant-adiabaticity radiofrequency pulses for generating long-lived singlet spin states in NMR.

A method is implemented to perform "fast" adiabatic variation of the spin Hamiltonian by imposing the constant adiabaticity condition. The method is applied to improve the performance of singlet-state Nuclear Magnetic Resonance (NMR) experiments, specifically, for efficient generation and readout of the singlet spin order in coupled spin pairs by applying adiabatically ramped RF-fields. Test experiments have been performed on a specially designed molecule having two strongly coupled 13C spins and on selectively isotopically labelled glycerol having two pairs of coupled protons. Optimized RF-ramps show improved performance in comparison, for example, to linear ramps. We expect that the methods described here are useful not only for singlet-state NMR experiments but also for other experiments in magnetic resonance, which utilize adiabatic variation of the spin Hamiltonian.

Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors.

BACKGROUND/AIMS: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. METHODS: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. RESULTS: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. CONCLUSION: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

Synthesis of plasmodione metabolites and 13C-enriched plasmodione as chemical tools for drug metabolism investigation.

Malaria is a tropical parasitic disease threatening populations in tropical and sub-tropical areas. Resistance to antimalarial drugs has spread all over the world in the past 50 years, thus new drugs are urgently needed. Plasmodione (benzylmenadione series) has been identified as a potent antimalarial early lead drug, acting through a redox bioactivation on asexual and young sexual blood stages. To investigate its metabolism, a series of plasmodione-based tools, including a fully 13C-labelled lead drug and putative metabolites, have been designed and synthesized for drug metabolism investigation. Furthermore, with the help of UHPLC-MS/MS, two of the drug metabolites have been identified from urine of drug-treated mice.

Complete magnetic field dependence of SABRE-derived polarization.

Signal amplification by reversible exchange (SABRE) is a promising hyperpolarization technique, which makes use of spin-order transfer from parahydrogen (the H2 molecule in its singlet spin state) to a to-be-polarized substrate in a transient organometallic complex, termed the SABRE complex. In this work, we present an experimental method for measuring the magnetic field dependence of the SABRE effect over an ultrawide field range, namely, from 10 nT to 10 T. This approach gives a way to determine the complete magnetic field dependence of SABRE-derived polarization. Here, we focus on SABRE polarization of spin-1/2 hetero-nuclei, such as 13 C and 15 N and measure their polarization in the entire accessible field range; experimental studies are supported by calculations of polarization. Features of the field dependence of polarization can be attributed to level anticrossings in the spin system of the SABRE complex. Features at magnetic fields of the order of 100 nT-1 µT correspond to "strong coupling" of protons and hetero-nuclei, whereas features found in the mT field range stem from "strong coupling" of the proton system. Our approach gives a way to measuring and analyzing the complete SABRE field dependence, to probing NMR parameters of SABRE complexes and to optimizing the polarization value.

cis Versus trans-Azobenzene: Precise Determination of NMR Parameters and Analysis of Long-Lived States of 15N Spin Pairs.

We provide a detailed evaluation of nuclear magnetic resonance (NMR) parameters of the cis- and trans-isomers of azobenzene (AB). For determining the NMR parameters, such as proton-proton and proton-nitrogen J-couplings and chemical shifts, we compared NMR spectra of three different isotopomers of AB: the doubly 15N labeled azobenzene, 15N,15N'-AB, and two partially deuterated AB isotopomers with a single 15N atom. For the total lineshape analysis of NMR spectra, we used the recently developed ANATOLIA software package. The determined NMR parameters allowed us to optimize experiments for investigating singlet long-lived spin states (LLSs) of 15N spin pairs and to measure LLS lifetimes in cis-AB and trans-AB. Magnetization-to-singlet-to-magnetization conversion has been performed using the SLIC and APSOC techniques, providing a degree of conversion up to 17 and 24% of the initial magnetization, respectively. Our approach is useful for optimizing the performance of experiments with singlet LLSs; such LLSs can be exploited for preserving spin hyperpolarization, for probing slow molecular dynamics, slow chemical processes and also slow transport processes.

The conformation of the Congo-red ligand bound to amyloid fibrils HET-s(218-289): a solid-state NMR study.

We have previously shown that Congo red (CR) binds site specifically to amyloid fibrils formed by HET-s(218-289) with the long axis of the CR molecule almost parallel to the fibril axis. HADDOCK docking studies indicated that CR adopts a roughly planar conformation with the torsion angle ϕ characterizing the relative orientation of the two phenyl rings being a few degrees. In this study, we experimentally determine the torsion angle ϕ at the center of the CR molecule when bound to HET-s(218-289) amyloid fibrils using solid-state NMR tensor-correlation experiments. The method described here relies on the site-specific 13C labeling of CR and on the analysis of the two-dimensional magic-angle spinning tensor-correlation spectrum of 13C2-CR. We determined the torsion angle ϕ to be 19°.

Thiolate Spin Population of Type I Copper in Azurin Derived from 33S Hyperfine Coupling.

The electron transfer mediating properties of type I copper proteins stem from the intricate ligand coordination sphere of the Cu ion in their active site. These redox properties are in part due to unusual cysteine thiol coordination, which forms a highly covalent copper-sulfur (Cu-S) bond. The structure and electronic properties of type I copper have been the subject of many experimental and theoretical studies. The measurement of spin delocalization of the Cu(II) unpaired electron to neighboring ligands provides an elegant experimental way to probe the fine details of the electronic structure of type I copper. To date, the crucial parameter of electron delocalization to the sulfur atom of the cysteine ligand has not been directly determined experimentally. We have prepared 33S-enriched azurin and carried out W-band (95 GHz) electron paramagnetic resonance (EPR) and electron-electron double resonance detected NMR (EDNMR) measurements and, for the first time, recorded the 33S nuclear frequencies, from which the hyperfine coupling and the spin population on the sulfur of the thiolate ligand were derived. The overlapping 33S and 14N EDNMR signals were resolved using a recently introduced two-dimensional correlation technique, 2D-EDNMR. The 33S hyperfine tensor was determined by simulations of the EDNMR spectra using 33S hyperfine and quadrupolar tensors predicted by QM/MM DFT calculations as starting points for a manual spectral fit procedure. To reach a reasonable agreement with the experimental spectra, the 33S hyperfine principal value, Az, and one of the corresponding Euler angles had to be modified. The final values obtained gave an experimentally determined sulfur spin population of 29.8 ± 0.7%, significantly improving the wide range of 29-62% reported in the literature. Our direct, experimentally derived value now provides an important constraint for further theoretical work aimed at unravelling the unique electronic properties of this site.

Coherent manipulation of non-thermal spin order in optical nuclear polarization experiments.

Time resolved measurements of Optical Nuclear Polarization (ONP) have been performed on hyperpolarized triplet states in molecular crystals created by light excitation. Transfer of the initial electron polarization to nuclear spins has been studied in the presence of radiofrequency excitation; the experiments have been performed with different pulse sequences using different doped molecular systems. The experimental results clearly demonstrate the dominant role of coherent mechanisms of spin order transfer, which manifest themselves in well pronounced oscillations. These oscillations are of two types, precessions and nutations, having characteristic frequencies, which are the same for the different molecular systems and the pulse sequences applied. Hence, precessions and nutations constitute a general feature of polarization transfer in ONP experiments. In general, coherent manipulation of spin order transfer creates a powerful resource for improving the performance of the ONP method, which paves the way to strong signal enhancement in nuclear magnetic resonance.

Anhydrous octyl-glucoside phase transition from lamellar to isotropic induced by electric and magnetic fields.

A static deuterium nuclear magnetic resonance (2HNMR) technique (magnetic field, B = 7.05 T) was employed to monitor the thermotropic lamellar phase of the anhydrous 1:1 mixture sample of octyl-b-D-glucoside (ßOG) and that of partially deuterium labelled at the alpha position on the chain, i.e.,ßOG-d2 In the absence of an electric field, the 2H NMR spectrum of the mixture gives a typical quadrupolar doublet representing the aligned lamellar phase. Upon heating to beyond the clearing temperature at 112 °C, this splitting converts to a single line expected for an isotropic phase. Simultaneous application of magnetic and electric fields (E = 0.4 MV/m) at 85 °C in the lamellar phase, whose direction was set to be parallel or perpendicular to the magnetic field, resulted in the change of the doublet into a single line and this recovers to the initial doublet with time for both experimental geometries. This implies E- and B-field-induced phase transitions from the lamellar to an isotropic phase and a recovery to the lamellar phase again with time. Moreover, these phase transformations are accompanied by a transient current. A similar observation was made in a computational study when an electric field was applied to a water cluster system. Increasing the field strength distorts the water cluster and weakens its hydrogen bonds leading to a structural breakdown beyond a threshold field-strength. Therefore, we suggest the observed field-induced transition is likely due to a structure change of the ßOG lamellar assembly caused by the field effect and not due to Joule heating.

Victor P. Whittaker (1919-2016).

This Obituary honors Victor P. Whittaker, one of the pioneers in the field of neurochemistry. Victor Whittaker died on 5th July 2016 aged 97 in Cambridge (UK) after a short illness. Victor is best known for his landmark advances in the subcellular fractionation of brain tissue which led to the isolation of synaptosomes and subsequently synaptic vesicles at the beginning of the 1960s and for the cellular and molecular analysis of the cholinergic synapse.

Melatonin receptor deficiency decreases and temporally shifts ecto-5'-nucleotidase mRNA levels in mouse prosencephalon.

Ecto-5'-nucleotidase (eN) is the major extracellular adenosine-producing ecto-enzyme in mouse brain. Via the production of adenosine, eN participates in many physiological and pathological processes, such as wakefulness, inflammation, nociception and neuroprotection. The mechanisms regulating the expression of eN are therefore of considerable neurobiological and clinical interest. Having previously described a modulatory effect of melatonin in the regulation of eN mRNA levels, we decided to analyze the melatonin receptor subtype involved in the regulation of eN mRNA levels by comparing eN mRNA patterns in melatonin-proficient transgenic mice lacking either the melatonin receptor subtype 1 (MT1 KO) or both melatonin receptor subtypes (MT1 and MT2; MT1/2 KO) with the corresponding melatonin-proficient wild-type (WT) controls. By means of radioactive in situ hybridization, eN mRNA levels were found to be diminished in both MT1 and MT1/2 KO mice compared with WT controls suggesting stimulatory impacts of melatonin receptors on eN mRNA levels. Whereas eN mRNA levels increased during the day and peaked at night in WT and MT1 KO mice, eN mRNA levels at night were reduced and the peak was shifted toward day-time in double MT1/2 KO mice. These data suggest that the MT2 receptor subtype may play a role in the temporal regulation of eN mRNA availability. Notably, day-time locomotor activity was significantly higher in MT1/2 KO compared with WT mice. Our results suggest melatoninergic signaling as an interface between the purinergic system and the circadian system.

NTPDase2 and purinergic signaling control progenitor cell proliferation in neurogenic niches of the adult mouse brain.

Nerve cells are continuously generated from stem cells in the adult mammalian subventricular zone (SVZ) and hippocampal dentate gyrus. We have previously noted that stem/progenitor cells in the SVZ and the subgranular layer (SGL) of the dentate gyrus express high levels of plasma membrane-bound nucleoside triphosphate diphosphohydrolase 2 (NTPDase2), an ectoenzyme that hydrolyzes extracellular nucleoside diphosphates and triphosphates. We inferred that deletion of NTPDase2 would increase local extracellular nucleoside triphosphate concentrations perturbing purinergic signaling and boosting progenitor cell proliferation and neurogenesis. Using newly generated mice globally null for Entpd2, we demonstrate that NTPDase2 is the major ectonucleotidase in these progenitor cell-rich areas. Using BrdU-labeling protocols, we have measured stem cell proliferation and determined long-term survival of cell progeny under basal conditions. Brains of Entpd2 null mice revealed increased progenitor cell proliferation in both the SVZ and the SGL. However, this occurred without noteworthy alterations in long-term progeny survival. The hippocampal stem cell pool and the pool of the intermediate progenitor type-2 cells clearly expanded. However, substantive proportions of these proliferating cells were lost during expansion at around type-3 stage. Cell loss was paralleled by decreases in cAMP response element-binding protein phosphorylation in the doublecortin-positive progenitor cell population and by an increase in labeling for activated caspase-3 levels. We propose that NTPDase2 has functionality in scavenging mitogenic extracellular nucleoside triphosphates in neurogenic niches of the adult brain, thereby acting as a homeostatic regulator of nucleotide-mediated neural progenitor cell proliferation and expansion.

Understanding the twist-bend nematic phase: the characterisation of 1-(4-cyanobiphenyl-4'-yloxy)-6-(4-cyanobiphenyl-4'-yl)hexane (CB6OCB) and comparison with CB7CB.

The synthesis and characterisation of the nonsymmetric liquid crystal dimer, 1-(4-cyanobiphenyl-4'-yloxy)-6-(4-cyanobiphenyl-4'-yl)hexane (CB6OCB) is reported. An enantiotropic nematic (N)-twist-bend nematic (NTB) phase transition is observed at 109 °C and a nematic-isotropic phase transition at 153 °C. The NTB phase assignment has been confirmed using polarised light microscopy, freeze fracture transmission electron microscopy (FFTEM), (2)H-NMR spectroscopy, and X-ray diffraction. The effective molecular length in both the NTB and N phases indicates a locally intercalated arrangement of the molecules, and the helicoidal pitch length in the NTB phase is estimated to be 8.9 nm. The surface anchoring properties of CB6OCB on a number of aligning layers is reported. A Landau model is applied to describe high-resolution heat capacity measurements in the vicinity of the NTB-N phase transition. Both the theory and heat capacity measurements agree with a very weak first-order phase transition. A complementary extended molecular field theory was found to be in suggestive accord with the (2)H-NMR studies of CB6OCB-d2, and those already known for CB7CB-d4. These include the reduced transition temperature, TNTBN/TNI, the order parameter of the mesogenic arms in the N phase close to the NTB-N transition, and the order parameter with respect to the helix axis which is related to the conical angle for the NTB phase.